Mark R. Holt

Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA

Directed cell migration is crucial for development, but most of our current knowledge is derived from in vitro studies. We analyzed how neural crest (NC) cells migrate in the direction of their target during embryonic development. We show that the proteoglycan Syndecan-4 (Syn4) is expressed in the migrating neural crest of Xenopus and zebrafish embryos. Loss-of-function studies using an antisense morpholino against syn4 show that this molecule is required for NC migration, but not for NC induction. Inhibition of Syn4 does not affect the velocity of cell migration, but significantly reduces the directional migration of NC cells. Furthermore, we show that Syn4 and PCP signaling control the directional migration of NC cells by regulating the direction in which the cell protrusions are generated during migration. Finally, we perform FRET analysis of Cdc42, Rac and RhoA in vitro and in vivo after interfering with Syn4 and PCP signaling. This is the first time that FRET analysis of small GTPases has been performed in vivo. Our results show that Syn4 inhibits Rac activity, whereas PCP signaling promotes RhoA activity. In addition, we show that RhoA inhibits Rac in NC cells. We present a model in which Syn4 and PCP control directional NC migration by, at least in part, regulating membrane protrusions through the regulation of small GTPase activities.

Focal adhesion kinase controls actin assembly via a FERM-mediated interaction with the Arp2/3 complex.

Networks of actin filaments, controlled by the Arp2/3 complex, drive membrane protrusion during cell migration. How integrins signal to the Arp2/3 complex is not well understood. Here, we show that focal adhesion kinase (FAK) and the Arp2/3 complex associate and colocalize at transient structures formed early after adhesion. Nascent lamellipodia, which originate at these structures, do not form in FAK-deficient cells, or in cells in which FAK mutants cannot be autophosphorylated after integrin engagement. The FERM domain of FAK binds directly to Arp3 and can enhance Arp2/3-dependent actin polymerization. Critically, Arp2/3 is not bound when FAK is phosphorylated on Tyr 397. Interfering peptides and FERM-domain point mutants show that FAK binding to Arp2/3 controls protrusive lamellipodia formation and cell spreading. This establishes a new function for the FAK FERM domain in forming a phosphorylation-regulated complex with Arp2/3, linking integrin signalling directly with the actin polymerization machinery.

Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band – implications for hereditary myopathies

Obscurin, a giant modular muscle protein implicated in G-protein and protein-kinase signalling, can localize to both sarcomeric Z-disks and M-bands. Interaction of obscurin with the Z-disk is mediated by Z-disk titin. Here, we unravel the molecular basis for the unusual localization of obscurin, a Z-disk-associated protein, to the M-band, where its invertebrate analogue UNC-89 is also localized. The first three domains of the N-terminus of obscurin bind to the most C-terminal domain of M-band titin, as well as to the M-band protein myomesin. Both proteins also interact with the N-terminal domains of obscurin-like 1 (Obsl1), a small homologue of obscurin. Downregulation of myomesin by siRNA interference disrupts obscurin–M-band integration in neonatal cardiomyocytes, as does overexpression of the binding sites on either myomesin, obscurin or Obsl1. Furthermore, all titin mutations that have been linked to limb-girdle muscular dystrophy 2J (LGMD2J) or Salih myopathy weaken or abrogate titin-obscurin and titin-Obsl1 binding, and lead to obscurin mislocalization, suggesting that interference with the interaction of these proteins might be of pathogenic relevance for human disease.

PTEN couples Sema3A signalling to growth cone collapse

Distinct changes in glycogen synthase kinase-3 (GSK-3) signalling can regulate neuronal morphogenesis including the determination and maintenance of axonal identity, and are required for neurotrophin-mediated axon elongation. In addition, we have previously shown a dependency on GSK-3 activation in the semaphorin 3A (Sema3A)-mediated growth-cone-collapse response of sensory neurons. Regulation of GSK-3 activity involves the intermediate signalling lipid phosphatidylinositol 3,4,5-trisphosphate, which can be modulated by phosphatidylinositol 3-kinase (PI3K) and the tumour suppressor PTEN. We report here the involvement of PTEN in the Sema3A-mediated growth cone collapse. Sema3A suppresses PI3K signalling concomitant with the activation of GSK-3, which depends on the phosphatase activity of PTEN. PTEN is highly enriched in the axonal compartment and the central domain of sensory growth cones during axonal extension, where it colocalises with microtubules. Following exposure to Sema3A, PTEN accumulates rapidly at the growth cone membrane suggesting a mechanism by which PTEN couples Sema3A signalling to growth cone collapse. These findings demonstrate a dependency on PTEN to regulate GSK-3 signalling in response to Sema3A and highlight the importance of subcellular distributions of PTEN to control growth cone behaviour.

Diminished sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca2+ in the cytosol ([Ca2+]i). A rise in [Ca2+]i is normalized by rapid reuptake of Ca2+ into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca2+ (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca2+]i, cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca2+]i following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca2+ re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca]i after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.

Vinculin acts as a sensor in lipid regulation of adhesion-site turnover

The dynamics of cell adhesion sites control cell morphology and motility. Adhesion-site turnover is thought to depend on the local availability of the acidic phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 can bind to many cell adhesion proteins such as vinculin and talin, but the consequences of this interaction are poorly understood. To study the significance of phospholipid binding to vinculin for adhesion-site turnover and cell motility, we constructed a mutant, vinculin-LD, deficient in acidic phospholipid binding yet with functional actin-binding sites. When expressed in cells, vinculin-LD was readily recruited to adhesion sites, as judged by fluorescence recovery after photobleaching (FRAP) analysis, but cell spreading and migration were strongly impaired, and PIP2-dependent disassembly of adhesions was suppressed. Thus, PIP2 binding is not essential for vinculin activation and recruitment, as previously suggested. Instead, we propose that PIP2 levels can regulate the uncoupling of adhesion sites from the actin cytoskeleton, with vinculin functioning as a sensor.

WIP Regulates the Stability and Localization of WASP to Podosomes in Migrating Dendritic Cells

Summary The Wiskott-Aldrich Syndrome protein (WASP) is an adaptor protein that is essential for podosome formation in hematopoietic cells [1]. Given that 80% of identified Wiskott-Aldrich Syndrome patients result from mutations in the binding site for WASP-interacting-protein (WIP) [2], we examined the possible role of WIP in the regulation of podosome architecture and cell motility in dendritic cells (DCs). Our results show that WIP is essential both for the formation of actin cores containing WASP and cortactin and for the organization of integrin and integrin-associated proteins in circular arrays, specific characteristics of podosome structure. We also found that WIP is essential for the maintenance of the high turnover of adhesions and polarity in DCs. WIP exerts these functions by regulating calpain-mediated cleavage of WASP and by facilitating the localization of WASP to sites of actin polymerization at podosomes. Taken together, our results indicate that WIP is critical for the regulation of both the stability and localization of WASP in migrating DCs and suggest that WASP and WIP operate as a functional unit to control DC motility in response to changes in the extracellular environment.

Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells

FLAP is a new method for localized photo‐labelling and subsequent tracking of specific molecules within living cells. It is simple in principle, easy to implement and has a wide potential application. The molecule to be located carries two fluorophores: one to be photobleached and the other to act as a reference label. Unlike the related methods of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), the use of a reference fluorophore permits the distribution of the photo‐labelled molecules themselves to be tracked by simple image differencing. In effect, FLAP is therefore comparable with methods of photoactivation. Its chief advantage over the method of caged fluorescent probes is that it can be used to track chimaeric fluorescent proteins directly expressed by the cells. Although methods are being developed to track fluorescent proteins by direct photoactivation, these still have serious drawbacks. In order to demonstrate FLAP, we have used nuclear microinjection of cDNA fusion constructs of β‐actin with yellow (YFP) and cyan (CFP) fluorescent proteins to follow both the fast relocation dynamics of monomeric (globular) G‐actin and the much slower dynamics of filamentous F‐actin simultaneously in living cells.

Structural insight into M-band assembly and mechanics from the titin-obscurin-like-1 complex

In the sarcomeric M-band, the giant ruler proteins titin and obscurin, its small homologue obscurin-like-1 (obsl1), and the myosin cross-linking protein myomesin form a ternary complex that is crucial for the function of the M-band as a mechanical link. Mutations in the last titin immunoglobulin (Ig) domain M10, which interacts with the N-terminal Ig-domains of obscurin and obsl1, lead to hereditary muscle diseases. The M10 domain is unusual not only in that it is a frequent target of disease-linked mutations, but also in that it is the only currently known muscle Ig-domain that interacts with two ligands—obscurin and obsl1—in different sarcomeric subregions. Using x-ray crystallography, we show the structural basis for titin M10 interaction with obsl1 in a novel antiparallel Ig-Ig architecture and unravel the molecular basis of titin-M10 linked myopathies. The severity of these pathologies correlates with the disruption of the titin-obsl1/obscurin complex. Conserved signature residues at the interface account for differences in affinity that direct the cellular sorting in cardiomyocytes. By engineering the interface signature residues of obsl1 to obscurin, and vice versa, their affinity for titin can be modulated similar to the native proteins. In single-molecule force-spectroscopy experiments, both complexes yield at forces of around 30 pN, much lower than those observed for the mechanically stable Z-disk complex of titin and telethonin, suggesting why even moderate weakening of the obsl1/obscurin-titin links has severe consequences for normal muscle functions.

Developmental regulation of MURF ubiquitin ligases and autophagy proteins nbr1, p62/SQSTM1 and LC3 during cardiac myofibril assembly and turnover

The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as ubiquitin E3 ligases in ubiquitin-mediated muscle protein turnover. Despite their well-characterised roles in muscle atrophy, the dynamics of MURF expression in the development and early postnatal adaptation of striated muscle is largely unknown. Here, we show that MURF2 is expressed at the very onset of mouse cardiac differentiation at embryonic day 8.5, and represents a sensitive marker for differentiating myocardium. During cardiac development, expression shifts from the 50 kDa to the 60 kDa A-isoform, which dominates postnatally. In contrast, MURF1 shows strong postnatal upregulation and MURF3 is not significantly expressed before birth. MURF2 expression parallels that of the autophagy-associated proteins LC3, p62/SQSTM1 and nbr1. SiRNA knockdown of MURF2 in neonatal rat cardiomyocytes disrupts posttranslational microtubule modification and myofibril assembly, and is only partly compensated by upregulation of MURF3 but not MURF1. Knockdown of both MURF2 and MURF3 severely disrupts the formation of ordered Z- and M-bands, likely by perturbed tubulin dynamics. These results suggest that ubiquitin-mediated protein turnover and MURF2 in particular play an unrecognised role in the earliest steps of heart muscle differentiation, and that partial complementation of MURF2 deficiency is afforded by MURF3.